Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of Avena sativa Phototropin 1

Abstract

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A′α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A′α helix of the LOV2 domain of Avena sativa phototropin 1 ( As LOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A′α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A′α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A′α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

“…8 This appears to stabilize the interaction between A’α and Jα, which was previously shown by Zayner and coworkers to be important for the unfolding of the Jα helix as observed by FTIR. 28 The TRIR and NMR spectra show that N414 is responsible for stabilizing the Jα helix in the dark state and that this helix is partially unfolded in the N414A mutant which has a critical impact on LOV domain function since the N414A LOVTRAP mutant has lost the ability to interact with the Zdk2 peptide in a light dependent manner.…”

“…A marker band for unfolding of the Jα helix was identified in the amide I region, ∼1620-1640 cm −1 using difference FTIR. 26–28 TRIR experiments revealed that helix unfolding was multi-step, 29 and that structural changes resulting from adduct formation follow dispersive kinetics commonly associated with sampling of multiple protein conformations prior to reaching a metastable state, which is the signaling state in LOV domain proteins. 30 TRIR spectroscopy of multiple LOV domain proteins revealed variations in the dynamics of the β-sheet and the Jα helix, which link the LOV domain to the relevant effector domains.…”

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

“…However, the A390 EADS curve resembles the steady-state spectrum of EL222 obtained under continuous illumination 32 and therefore contains changes in protein secondary, tertiary and possibly quaternary structure (oligomerisation). More specifically, unfolding of the A 0 a-helix, 57,90 unfolding of Ja-helix, 28,88,91 and rearrangement of beta-sheets 90 have been postulated in the spectral region between 1620 and 1670 cm À1 of many LOV domains. While the aforementioned large-scale effects are not incorporated in the current computational setup, the good agreement of the computed Qc pertains mostly to features above baseline, hinting that quite a few of those features can be assignable to small-scale effects.…”

“…The key intermediates along the photocycle were modelled and analysed vibrationally, producing difference spectra, which were then correlated with the experimental infrared spectra of the EL222 and AsLOV2 proteins from this work and the literature. 7,32,57 Since both the photocycle and the spectroscopic analysis are presented here through the prism of the key glutamine residue, the possibility of imidic tautomerisation of its side chain was also considered for the LOV domains (Scheme 1). This was highlighted before in BLUF, 37,53,[58][59][60][61] a class of blue-light absorbing photoreceptors sharing the isoalloxazine ring.…”

Characterization of a Vivid Homolog in Botrytis cinerea

“…Importantly, these studies are performed in truncated proteins that may lead to artifacts due to construct length. Indeed, recent studies of AsLOV2 have demonstrated that the N‐terminal A′α helix is required for light‐driven Jα unfolding, and constructs containing A′α extensions can attenuate conformational changes in the Jα helix .…”